Aldehyde dehydrogenases (ALDHs) are intracellular enzymes responsible for oxidizing aldehydes. Substrates for ALDHs include acetyldehyde, an intermediate in ethanol metabolism, and biogenic amines produced during catecholamine catabolism. (Russo et al., Cancer Res. 48: 2963-2968 (1988)). ALDH has also been reported to play a crucial role in the conversion of vitamin A to its active metabolite, retinoic acid (Labrecque et al., Biochem. Cell Biol. 71:85-89 (1993); Yoshida et al., Enzyme 46:239-244 (1992)).
High enzymatic activity of ALDH has been shown to be a characteristic feature of primitive hematopoietic progenitor cells in mice and humans (Kohn et al., Biochem. Pharmacol. 34:3465-3471 (1985); Kastan et al., Blood 75:1947-1950 (1990)). ALDH activity has been used as a marker to identify and enrich hematopoietic stem cells (Jones et al., Blood, 85(10): 2742-2746 (1995)), and to assess the quality of cells as hematopoietic stem cell transplants (Lioznov et al., Bone Marrow Transplant., 35:909-914 (2005)). An assay based on ALDH activity useful in enumerating endogenous progenitor cells in peripheral blood has been described (Povsic et al., J. Am. Coll. Cardiol., 50:2243-2248 (2007)).
Oxazaphosphorines (e.g., cyclophosphamide) are latent drugs that provide a chemically and pharmacologically inactive form of nitrogen mustards, cytotoxic chemotherapeutic agents. Oxazaphosphorines are metabolized to their active forms in vivo. For example, cyclophosphamide is a prodrug that requires metabolic activation to exhibit cytotoxic activity. The primary metabolite of cyclophosphamide, 4-hydroxycyclophosphamide (4-OH-CPA), is in equilibrium with its open-ring tautomer, aldophosphamide, which undergoes chemical decomposition to form phosphoramide mustard (a bifunctional DNA alkylator) and acrolein, with phosphoramide mustard being the ultimate cytotoxic metabolite. Alternatively, 4-OH-CPA and aldophosphamide are detoxified by glutathione S-transferase with thiols or sulfates and by ALDH to carboxycyclophosphamide, respectively. (Brock, Cancer, 78(3): 542-47 (1996)).
The dosage of oxazaphosphorines such as cyclophosphamide, and their toxicity profiles, vary widely depending on the clinical indication. Fifty years after it was first synthesized, cyclophosphamide continues to be used for a wide array of diseases, including solid tumors, hematologic malignancies, autoimmune disorders, stem cell mobilization, and as a conditioning regimen for bone marrow transplant (Emadi et al., Nat. Rev. Clin. Oncol., 6:638-647 (2009)).
The chemotherapeutic properties of oxazaphosphorines have been demonstrated in a wide range of tumors. (Brock, Cancer, 78(3): 542-47 (1996)). For example, cyclophosphamide is one of the few drugs with a broad indication for cancer, and has been included in various chemotherapeutic regimens, as a monotherapy or in combination with other anti-neoplastic drugs (for example, 40-50 mg/kg over a period of 2-5 days, 10 to 15 mg/kg every 7-10 days, or 3-5 mg/kg twice weekly). Moreover, high-dose cyclophosphamide (for example, 50 mg/kg/day×4 days) has been used for the treatment of certain autoimmune diseases such as, for example, severe aplastic anemia. High-dose cyclophosphamide was originally used in allogeneic bone marrow transplantation because of its ability to break immune tolerance and facilitate engraftment. (Santos et al., Transplant Proc., 4: 559-564 (1972)).
It has been observed that high levels of cytosolic ALDH produce cyclophosphamide-resistance in tumor cell lines (Russo J E and Hilton, J, Cancer Res., 48:2963 (1988)). It has been shown that measurable levels of some ALDH enzymes are found in some, but not all, tumor types. Furthermore, in those tumor types where measurable ALDH levels are present (e.g., carcinomas of the breast), inter-individual variation may exist (ALDH levels may vary from patient to patient). Further, it has been proposed that ALDH-1 and ALDH-3 levels/activities in tumors can be used to predict the therapeutic potential of oxazaphosphorine chemotherapy regimens, e.g., in breast cancer (Sreerama L and Sladek N E, Cancer Res., 54:2176-2185 (1994); Sladek N E, Curr. Pharm. Des., 5(8):607-625 (1999); Sladek N E et al., Cancer Chemother. Pharmacol., 49(4):309-321 (2002)). Other proposed clinical strategies based on ALDH include: sensitizing tumor cells to oxazaphosphorines by inhibiting synthesis of ALDH or ALDH activity; and decreasing the sensitivity of vulnerable and essential normal cells, such as pluripotent hematopoietic cells, to oxazaphosphorines by increasing ALDH1 or ALDH3 through gene delivery (Sladek N E et al., 2002).